Field flow fractionation is a versatile technique for the high resolution separation of a wide variety of particulates, including both particles and macromolecules, suspended in a fluid medium. The particulates include macromolecules in the 10.sup.5 to the 10.sup.13 molecular weight (0.001 to 1 .mu.m) range, colloids, particles, unicelles, organelles and the like. The technique is more explicitly described in U.S. Pat. No. 3,449,938, issued June 17, 1969 to John C. Giddings and U.S. Pat. No. 3,523,610, issued Aug. 11, 1970 to Edward M. Purcell and Howard C. Berg.
Field flow fractionation is the result of the differential migration rate of components in a carrier or mobile phase in a manner similar to that experienced in chromatography. However, in field flow fractionation there is no separate stationary phase as is in the case of chromatography. Sample retention is caused by the redistribution of sample components between the fast to the slow moving strata within the mobile phase. Thus, particulates elute more slowly than the solvent front. Typically, a field flow fractionation channel consisting of two closely spaced parallel surfaces is used. A mobile phase is caused to flow continuously through the gap between the surfaces. Because of the narrowness of this gap or channel (typically 0.025 centimeters (cm)) the mobile phase flow is laminar with a characteristic parabolic velocity profile. The flow velocity is the highest at the middle of the channel and the lowest near the two channel surfaces.
An external influencing or force field of some type (the force fields include gravitational, thermal, electrical, fluid cross-flow and others as described variously by Giddings and Berg and Purcell), is applied transversely (perpendicular) to the channel surfaces or walls. This force field pushes the sample components in the direction of the slower moving strata near the outer wall. The buildup of sample concentration near the wall, however, is resisted by the normal diffusion of the particulates in a direction opposite to the force field. This results in a dynamic layer of component particles, each component with an exponential-concentration profile. The extent of retention is determined by the time-average position of the particulates within the concentration profile which is a function of the balance between the applied field strength and the opposing tendency of particles to diffuse.
In sedimentation field flow fractionation (SFFF), use is made of a centrifuge to establish the force field required for the separation. For this purpose a long, thin, annular belt-like channel is made to rotate within a centrifuge. The resultant centrifugal force causes components of higher density than the mobile phase to settle toward the outer wall of the channel. For equal particle density, because of their higher diffusion rate, smaller particulates will accumulate into a thicker layer against the outer wall than will larger particles. On the average, therefore, larger particulates are forced closer to the outer wall.
If now the fluid medium, which may be termed a mobile phase or solvent, is fed continuously in one end of the channel, it carries the sample components through the channel for later detection at the outlet of the channel. Because of the shape of the laminar velocity profile within the channel and the placement of particulates in that profile, solvent flow causes small particulates to elute first, followed by a continuous elution of sample components in the order of ascending particulate mass.
In a sedimentation field flow fractionation apparatus, with constant force field strength, particle retention is directly proportional to particulate mass and to the third power of particulate size. This fundamental relationship is described by Giddings et al. in a paper F. J. F. Yang, M. N. Myers, and J. C. Giddings, Analytical Chemistry, 46, 1924 (1974). Most SFFF separations have been carried out with a constant force field. Unfortunately, however, since SFFF retention in a constant field is linearly related to particulate mass, the dependence of retention time on particulate size is highly nonlinear. Hence, the conversion of a constant field SFFF fractogram to a sample particulate size distribution curve is inconvenient to say the least.
Further problems with constant field SFFF analysis or separations are the long times required to effect separation and the poor detection of late eluting species because of broad peaks. These problems are related to the fact that a constant field SFFF analysis does not exhibit constant resolution (separating power) across the desired wide particulate mass separation range. In constant field separations, the high field strength required to resolve small particulates invariably causes excessive retention of large particulates. In addition, late eluting large particulates are also badly dispersed (diluted) as they elute from the SFFF channel, causing detection problems.
Giddings et al. sought to reduce the long analysis time required and to alleviate the poor detectability resulting from constant field SFFF separations. They sought to do this by using step and linear field decay programs. Parabolic field programming of thermal gradients have also been used in thermal FFF. This is described in an article by J. C. Giddings et al., Analytical Chemistry, 48, 1587 (1976) entitled "Programmed Thermal Field-Flow Fractionation." Although these programming schemes improve the analysis time and sample detectability, they inadvertently create uncertainties in the quantitative relationship between retention and particle mass or particulate size. These programming schemes sacrifice the simple retention-mass relationship of constant field SFFF. It would also be highly desirable to provide SFFF separation techniques in which separation range and resolution could be varied, and at the same time a convenient retention-mass relationship could be maintained for easy and accurate determination of particulate size or molecular weights.
Giddings et al., in Analytical Chemistry, 46, 1917 (1974) noted that with increased flow rates, rapidly eluted components in field flow fractionation tend to merge into the void or solvent peak if high flow rates are used. Conversely if low flow rates are used, the more highly retained components are greatly delayed in their elution. Giddings et al. in Anal. Chem. 51, 30 (1979) suggest that the flow rate of the mobile phase may be increased in steps or by a simple proportional function to time raised to a power to alleviate some of these problems. Unfortunately, this method does not provide a convenient retention-mass (or field-affected particulate characteristic) relationship that is useful in analytical determinations.